Characterization of highly expressed miR-125b-5p in Japanese encephalitis virus persistently-infected cells

• Main Authors: Chih-Wei Huang, 黃至韡
• Other Authors: Ruey-Yi Chang
• Format: Others
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/08444143803172164143

碩士 === 國立東華大學 === 生命科學系 === 104 === A number of studies have been reported that host or virus-derived microRNAs (miRNAs) have participated versatile roles in multiple biological processes. How miRNAs may have involved in the establishment of persistent infection with flaviviruses remains mostly unknown. We have shown previously that Japanese encephalitis virus (JEV) could establish persistent infection in mammalian (BHK-21) cells. To characterize the potential roles of miRNAs in the establishment of persistent infection, small RNA deep sequencing approach was performed to obtain the miRNA expression profile in comparison between the acute and persistent infection. The results showed that miR-125b-5p is the most abundant expressed one in JEV persistently infected cells. In this study, I further characterized the abundance of miR-125b-5p by northern blot and quantitative RT-PCR. I demonstrated that as soon as the cells established persistent infection, the significant high expression of miR-125b-5p was readily observed. Transfecting of miR-125b-5p at low dosage (< 5 nM) slightly increased cell viability, while high concentration at 25 nM significantly reduced cell viability indicting that miR-125b-5p has dose effect. Transfecting of miR-125b-5p at 0.5 nM did not rescue virus-induced cell death but significantly reduced virus titers. Six potential targets of miR-125b-5p were predicted using bioinformatics tools. These targets are down regulated in persistently infected cells and are direct targets of miR-125b-5p as evidenced by luciferase reporter assay. Taken together, these results demonstrated that miR-125b-5p up regulated in persistently infected cells, inhibited virus replication presumably by targeting to several host regulators in signal transduction pathway that could lead to persistent infection. This is the first report that characterized potential roles of miRNAs in the establishment of flaviviral persistent infection.